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United States Patent |
5,637,453
|
Jehuda-Cohen
|
June 10, 1997
|
Whole blood method and kit for the detection of antibodies against human
immunodeficiency virus (HIV) in HIV-seronegative individuals
Abstract
The present invention relates to an improved method and kit for the
detection of antibodies directed against the human immunodeficiency virus
(HIV) in whole blood obtained from individuals who are HIV-seronegative as
determined by conventional assay techniques. Whole blood is isolated from
these individuals and cultured directly with mitogen to stimulate the
proliferation of B-lymphocytes producing immunoglobulin specific for HIV.
This method provides a rapid, simple, and inexpensive means for screening
large numbers of blood samples for HIV infection while minimizing exposure
of the technician to infectious blood components.
Inventors:
|
Jehuda-Cohen; Tamar (Rehovot, IL)
|
Assignee:
|
Shiloov Medical Technologies, Ltd. (Rehovot, IL)
|
Appl. No.:
|
275933 |
Filed:
|
July 15, 1994 |
Current U.S. Class: |
435/5; 435/7.1; 436/63; 436/536 |
Intern'l Class: |
C12Q 001/70; G01N 033/53; G01N 033/48; G01N 033/536 |
Field of Search: |
435/5,2.1,974,975
436/536,63,160.1,208.1
|
References Cited
Other References
Siekovitz et al, Science 238:1575-1578. 1987.
Israel et al, J. of Immunology, 143(12):3956-3960. 1989.
Bohnlein et al, Cell 53:827-836. 1988.
Cole et al, Abstract Int. Conf. Ans Jul. 1992 Ab#PoC4216.
Chowhurdy et al, Virology 176:126-132. 1990.
Tamar, Jehuda-Cohen et al "Polyclonal B-cell activation reveals antibodies
against human immunodeficiency virus type-1 (HIV-1) in HIV-l seronegative
individuals" PNAS. 87. May 1990. pp. 3972-3976.
Savita Pahwa et al "In vitro synthesis of human immunodeficiency virus
specific antibodies in peripheral blood lymphocytes of infants", PNAS, 86.
Oct. 1989. pp. 7532-7536.
R.H. Zubler et al "Frequencies of HIV-reactive B-cells in sepopositive and
seronegative individuals" Clin Exp. Immuno. 87. Jun. 1992. pp. 31-36.
Alberto Amadori et al. "HIV-1-Specific B Cell Activation". J. Imm. 143(7).
Oct. 1989. pp. 2146-2152.
Robert Yarchocn et al "Mechanisms of B cell Activation in Patients with
acquired Immuno-deficiency Syndrome and Related Disorders" J. Clin.
Invest. 78, Aug. 1986. pp. 434-447.
Hall and Gordon, 1976, "Reproducibility, efficacy, and methodology of
mitogen-induced lymphocyte transformations by the whole blood assay", J.
Immunol. Methods 12:31-38.
Strongin, W., 1993, "Sensitivity, Specificity, and Predictive Value of
Diagnostic Tests: Definitions and Clinical Applications", in Laboratory
Diagnosis of Viral Infections, Lennette, E., ed., Marcel Dekker, Inc., New
York, pp. 211-219.
DeFranco, A., 1993, "B Lymphocyte Activation" in Fundamental Immunology,
Paul, ed., Raven Press, Ltd., New York, p. 525.
|
Primary Examiner: Nucker; Christine M.
Assistant Examiner: Parkin; Jeffrey S.
Attorney, Agent or Firm: Cooper & Dunham LLP
Parent Case Text
This is a continuation, of application Ser. No. 08/095,824, filed Jul. 21,
1993, now abandoned, which is a continuation of application Ser. No.
07/797,730, filed Nov. 25, 1991.
Claims
I claim:
1. An in vitro method for the detection of antibodies directed against a
human immunodeficiency virus in a sample obtained from a subject,
comprising the following steps:
a) obtaining a whole blood sample from the subject;
b) incubating the whole blood sample in a culture in the presence of a
media containing mitogen, to induce polyclonal activation of peripheral
blood mononuclear cells and the expression of human immunodeficiency virus
specific antibodies;
c) exposing the resultant culture of step b) to an human immunodeficiency
virus antigen, thereby allowing an antigen-antibody immune complex to
form;
d) detecting the antigen-antibody immune complex of step c); wherein the
presence of human immunodeficiency virus specific antibodies is indicative
of the subject being exposed to human immunodeficiency virus.
2. The method of claim 1, wherein the culture of step b) results in a
supernatant, and the supernatant is exposed to an human immunodeficiency
virus antigen, thereby allowing an antigen-antibody immune complex to
form.
3. The method of claim 1, wherein the mitogen is pokeweed mitogen, lectins,
bacterial endotoxins, viruses, lipid A or lymphokines.
4. A kit for the detection of antibodies directed against a human
immunodeficiency virus from a subject, comprising: a container for
collecting whole blood samples, wherein the container contains a media
containing mitogen, effective to induce polyclonal activation of
peripheral blood mononclear cells and the expression of human
immunodefidency virus specific antibodies of the whole blood sample.
5. The kit of claim 4, wherein the kit additionally comprises an assay for
the detection of human immunodeficiency virus specific antibodies.
6. The kit of claim 5, wherein the assay is an enzyme linked immunosorbent
assay, a western blot, or an immunofluorescence assay.
7. The kit of claim 4, wherein the container is made of a plastic, glass,
or metal material.
8. The kit of claim 7, wherein the container is a test tube or a flask.
9. The kit of claim 4, wherein the container is vacuum sealed.
10. The kit of claim 9, wherein the container is a vacutube.
Description
TECHNICAL FIELD
The present invention relates to an improved method and kit for detecting
antibodies in whole blood of individuals who test seronegative by
conventional assay techniques. More particularly, the present invention
relates to an assay for detecting possible retrovirus infection, such as
infection by the HIV virus, which utilizes a mitogen in whole blood to
stimulate antibody production by peripheral blood mononuclear cells. The
present invention also relates to an improved assay kit which does not
require the separation of peripheral blood mononuclear cells from whole
blood prior to culture with pokeweed mitogen.
Background of the Invention
As used herein, mitogen means any substance capable of activating B-cells
and/or T-cells. The term "whole blood" means blood collected with heparin,
EDTA, or any other substance that prevents coagulation and clotting. The
term whole blood as used herein also includes blood collected from an
animal or human with heparin, ethylenediaminetetraacetate, or any other
substance that prevents coagulation and clotting. "Whole blood" can also
mean blood wherein the red blood cells have been lysed while maintaining
the viability of the remaining white blood cells.
Serological detection of antibodies against a variety of infectious disease
agents is considered evidence of exposure to and/or active infection by
the agent. Serological detection of antibodies could also be useful for
early detection of cancer and for predicting the success of organ or
tissue transplants. Enzyme-linked immunosorbent assay (ELISA) commercial
kits are commonly used as screening tests for serological detection of
antibodies. The western blot technique has been the method most widely
used to confirm ELISA-reactive serum samples, although other methods such
as immunofluorescence, may also be applicable. Polymerase chain reaction
(PCR) technique may also be used to confirm results of a preliminary
assay.
As part of standard ELISA procedure, test serum is incubated with specific
antigens that are immobilized on beads or wells. Non-specific antibody in
the serum is removed by washing, but the antibodies with affinity for the
antigens present in the system remain bound. When the appropriate
developing reagents are added, spectrophotometrically detectable color is
produced, the optical density of which is proportional to the amount of
antibodies bound. The standard optical density is established by the
manufacturer of the ELISA kit and affects both the sensitivity and
specificity of the assay. Generally, a sample that is positive is retested
twice and deemed "positive" only if at least one of the subsequent two
tests is also reactive. Due to its subjective nature, the prediction value
of a positive ELISA varies depending on the degree of ELISA reactivity and
the probability of infection. Additionally, results may be affected by the
presence of a variety of other conditions, including autoimmune disease.
Western blot technique is widely used to confirm ELISA-reactive serum
samples. In the Western blot test, the desired antigens are
electrophoretically separated into discrete bands that are then
transferred onto nitrocellulose paper. Particular antigens will exhibit
identifiable and characteristic banding patterns. The nitrocellulose test
strips are then incubated with donor serum specimens. Antibodies present
in the sample will bind with specific antigenic bands and thus facilitate
separation and identification of the antibodies present. Prepared
nitrocellulose test strips are commercially available for a variety of
tests. The Western blot technique is considered more specific than the
ELISA technique, yet it is usually less sensitive.
Current serological techniques, however, do not identify individuals who
are infected but lack detectable levels of reactive antibodies. Examples
of conditions in which detectable levels of reactive antibodies are
lacking include autoimmune diseases, where antibody may be present only a
portion of the time and suppressed the remainder of the time or where
antibodies are bound to the antigen forming immune complexes and thus may
be nondetectable in serum; some forms of cancer, where antibody production
against the tumor may be suppressed by some specific process in the
development of the cancer; organ and tissue transplants, where the
recipient is not producing antibodies against the potential donor but
would suffer rapid graft rejection because of recall stimulation of the
immune system due to a cross-reaction of the donor's antigens with
antigens the recipient was previously exposed to; cytomegalovirus, which
causes a reduction in antibody production; and, a host of other infections
in which antibody production is subsequently suppressed. A variety of
viruses can interfere with immunological functions as well. The inhibition
that is induced may be specifically related to immune reactions to the
virus or may be non-specific and affect many components of the general
immune system of the host.
For example, recently a new class of human retroviruses which infect a
subset of lymphocytes has been shown to cause profound immunological
suppression and to cause an individual who has been infected with the
virus to develop susceptibility to many pathogenic organism. Human
Immunodeficiency Virus (HIV) infects T-lymphocytes belonging to the helper
cell subset. The infection and subsequent loss of T-helper cells is
thought to lead to immunosuppression and the resulting acquired
immunodeficiency syndrome (AIDS).
AIDS was first reported by the Center for Disease Control (CDC) in 1981.
Individuals were defined as having AIDS if the following conditions were
present: (1) a reliably diagnosed disease such as P. Carinii pneumonia,
other opportunistic infection, or Kaposi's sarcoma in a person less than
60 years of age that suggested an underlying cellular immune defect, and
(2) occurrence of the disease in the absence of a cellular immune
deficiency that could be ascribed to another factor (Samter, M., ed.
"Immunological Diseases", 4th ed, p. 445 (1988)). Two related disorders
were also noted which manifested a variety of signs and symptoms
suggestive of AIDS but did not meet the criteria established by the CDC.
These syndromes are described by the terms AIDS-related complex (ARC) and
chronic lymphadenopathy. ARC is characterized by fatigue, fever, night
sweats, diarrhea, unintentional weight loss, oral candidiasis, generalized
lymphadenopathy, leukopenia, and anemia, accompanied by immunological
abnormalities similar to AIDS. Chronic lymphoadenapathy syndrome describes
a condition of chronic lymphadenopathy of at least 6 months duration and
affecting two or more extrainguinial sites in the absence of an illness or
drug use known to cause lymphadenopathy. "Immunological Diseases", supra.,
at p.445-446.
AIDS and its related syndromes are attributed to a lymphocytotrophic
retrovirus designated: human immunodeficiency virus (HIV). HIV can be
readily recovered from individuals with early stages of AIDS but cannot
always be recovered intact from individuals in the late stages of AIDS. It
is postulated that this is because the subset of T cells thought to harbor
the virus has been depleted.
Serological screening techniques are being utilized worldwide for the
detection of human immunodeficiency virus type 1 (HIV-1). The presence of
antibody against human immunodeficiency virus type 1 (HIV-1) is considered
a strong indicator of HIV-1 infection. An ELISA assay is currently being
utilized on serum samples in most hospitals and screening rooms to make
this determination. A similar assay is being used to detect the presence
of simian immunodeficiency virus (SIV), a virus similar to HIV found in
nonhuman primates. If the serum sample is positive, an aliquot of the
sample is screened by a Western blot assay kit for confirmation. The
presence of antibody against two to three of the major protein bands of
the virus is considered a positive confirmation and identification that
the serum sample donor is infected.
Experimental results indicate that the currently used ELISA assays do not
detect all HIV infected individuals. This is because some HIV infected
individuals do not have detectable levels of serum antibody to HIV-1 and
current techniques do not identify individuals who lack detectable levels
of HIV-1 antibodies. Studies indicate that there can be a considerable
time lag between detection of HIV-1 infection and seroconversion.
Additionally, some HIV infected but seronegative individuals might never
convert but will remain infected throughout their lives. Thus, there is a
significant number of false negatives being reported. The existence of
HIV-1 infected but seronegative individuals has been documented using the
polymerase chain reaction (PCR) technique, virus isolation techniques, and
in situ hybridization. Identifying infected but seronegative individuals
is critical to controlling the spread of the disease due to its highly
contagious nature. Additionally, data from such assays have important
consequences for the clinical management, follow-up, and therapy of
infected individuals as well as maintaining the safety of health care
workers who come into contact with infected individuals.
Recently a method for detecting HIV infection in seronegative individuals
was reported in the scientific literature. (Jehuda-Cohen, T., "Polyclonal
B-cell activation reveals antibodies against human immunodeficiency virus
type 1 (HIV-1) in HIV-1-seronegative individuals" Proc. Nat. Acad. Sci.
USA, Vol 87, pp. 3972-3076, 1990). In this article, a method is described
wherein peripheral blood mononuclear cells (PBMC) are isolated from the
blood and then exposed to a mitogen such as pokeweed mitogen. It was found
that in those patients which had been infected by HIV and were
seronegative, incubation of isolated PBMC with pokeweed mitogen caused the
PBMC to secrete immunoglobulins that were specific for HIV. Thus, this
test provides the possibility of identifying a significant portion of
those patients that have been infected with HIV but are seronegative.
However, there are some serious problems in using this test in the clinic.
First of all, the blood from the patient who has been exposed to HIV has
to be collected and then fractionated to isolate the peripheral blood
mononuclear cells. These cells then must be suspended in a growth medium
with a mitogen therein and incubated for an appropriate amount of time.
These procedures must be conducted under sterile conditions. The
supernatant is then tested for the presence of HIV specific antibody. The
process of maintaining a sterile environment is time-consuming and the
process of isolating and collecting PBMC exposes the technician to blood
that contains HIV. While this practice may be acceptable in the research
laboratory, in the clinical laboratory, where hundreds, and in some cases
thousands of blood samples are handled each week, the possibility of a
technician becoming infected by the blood is greatly increased.
What is needed in the art is an assay which allows detection of infection
by a particular antigen, such as the HIV virus, prior to seroconversion.
The assay should include minimal exposure to the blood by the technician
and, at the same time, provide a safe, easy and inexpensive method of
screening large numbers of blood samples for viral infection.
SUMMARY OF THE INVENTION
The present invention relates to an improved assay for detecting antibodies
in whole blood of individuals who test seronegative by conventional assay
techniques, thus aiding in the diagnosis of possible retrovirus
infections. More particularly, the present invention relates to an
improved assay and kit which utilizes a mitogen, such as pokeweed mitogen,
in whole blood to stimulate the production of antibodies previously
undetectable.
In accordance with the present invention, a blood sample is drawn into a
test tube, such as a vacutube, containing an effective concentration of a
solution of a mitogen, such as pokeweed mitogen. The blood sample to be
tested is cultured in vitro in the presence of the pokeweed mitogen. Other
activators of human B cells may be used in place of or in addition to the
pokeweed mitogen to achieve the same function. After incubation, an
aliquot is taken from the top of the fluid and is then assayed for the
presence of desired antibodies using standard ELISA procedures and/or
Western Blot analysis. If the sample is to be assayed at a later date, the
blood may be centrifuged and the supernatant fluid may be collected,
frozen and stored. Results may be verified utilizing the technique of
polymerase chain reaction (PCR).
Alternatively, PBMC's may be separated from the blood sample to be tested
and cultured alone with a mitogen, such as pokeweed mitogen, and in a
culture medium. PBMC alone may be used to detect "hidden" antibodies to
cancer epitopes or for matching donors for transplantations.
Accordingly, it is an object of the present invention to provide a simple
and sensitive assay for the detection of retrovirus infections in patients
who are infected but are seronegative for the virus.
It is another object of the present invention to provide a simple and
sensitive assay to be used for predicting the possibility of organ or
tissue rejection due to recall stimulation of antibody production in
transplant recipients.
It is another object of the present invention to provide a method and kit
for early detection of some forms of cancer.
It is another object of the present invention to provide a method for
diagnosing HIV infection.
It is another object of the present invention to provide an assay kit which
is self contained and does not require the separation of peripheral blood
mononuclear cells prior to assay.
It is another object of the present invention to provide an assay and assay
kit for diagnosis of retrovirus infections which decreases risk to the
health care workers by simplifying the assay procedure, reducing the
handling of the donor sample, and thereby reducing the risk of infection.
It is another object of the present invention to provide a means for
detecting antibodies when levels of antibodies in the blood are lower than
those currently detectable by conventional assay systems.
These and other objects, features and advantages of the present invention
will become apparent after a review of the following detailed description
of the disclosed embodiment and the appended claims.
Detailed Description of the Present Invention
The present invention comprises a method and kit for the detection of
"hidden" antibodies in whole blood of individuals who tested seronegative
by conventional assay techniques. Additionally, the invention comprises a
method for detecting HIV antibodies in whole blood of those individuals
who have been infected with HIV but appear seronegative with conventional
assay techniques. The method involves incubating whole blood of
seronegative individuals in the presence of a mitogen, such as pokeweed.
The mitogen causes activation of the peripheral blood mononuclear cells
and the production of antibodies. The presence of specific antibodies may
then be determined by the use of any conventional assay techniques such as
those outlined above.
In the present invention, any mitogen can be used to activate the cells.
The mitogen can be either T-cell dependent or T-cell independent. The
preferred mitogen is pokeweed mitogen. Other mitogens can be used in
practicing the present invention and include, but are not limited to,
lectins, such as, concanavalin A; bacterial endotoxins; bacterially
derived lipid A; a variety of viruses; and, biological agents such as
lymphokines, including, but not limited to, interleukin-4, interleukin-5
and interleukin-6, or other anti-immunoglobulin reagents. The optimal
concentration of mitogen is easily determined without undue
experimentation by one of ordinary skill in the art. With regard to the
preferred mitogen, pokeweed mitogen, the preferred concentration range is
between approximately 1:100 and 1:1600 dilutions of stock PWM. The most
preferred concentration range is between approximately 1:200 and 1:400
dilutions of stock PWM. The preferred source of the stock PWM is GIBCO,
New York, N.Y. The lyophilized PWM is reconstituted with 5 ml of distilled
water to make the stock solution. The estimated concentration range of
pokeweed mitogen is 1 to 100 .mu.g/ml.
Culture medium means any medium that can be used to practice the present
invention, including but not limited to RPMI 1640 (GIBCO, New York, N.Y.),
preferably supplemented with appropriate antibiotics and glutamine. Other
culture media which may be used in practicing the present invention
include, but are not limited to, Eagles, Dulbecco's , McCoy's , Media 199
and Waymouth's media.
The present invention also includes a kit comprising a blood collection
container containing an effective concentration of mitogen therein. The
container can optionally contain a culture medium. The preferred container
is a test tube. The blood collection container can be plastic, glass, or
any other material that is compatible with culturing blood. It is to be
understood that the present invention also includes blood containing means
other than a blood collection tube including, but not limited to,
microtiter plates containing wells in which the blood can be incubated,
tissue culture flasks, glass flasks such as an erlenmeyer flask, and any
other container in which the blood can be cultured
The method of the present invention includes optionally separating the
blood cells from the fluid portion of the blood so that the presence of
antibodies can be determined. The separation of the blood cells from the
fluid portion of the blood can be done by any of several methods well
known to those of ordinary skill in the art, including centrifugation or
filtration. It is to be understood, that the blood cells do not need to be
physically separated from the fluid. Although, in those situations where
risk of infection is low, PBMC's may be separated from the blood prior to
culture and assay. After incubation of the whole blood with the mitogen,
fluid from the top of the blood can easily be extracted and tested for
antibody. Optionally, the red blood cells can be lysed either by mild
osmotic shock or with a mild detergent. In this way, the white blood cells
remain viable.
In one embodiment of the present invention, whole blood is collected in a
blood collection tube containing culture medium and mitogen. The blood
samples are then incubated with an approximately 1:500 final dilution of
pokeweed mitogen at a concentration of 2.times.10.sup.6 viable cells per
ml for four days at 37.degree. C. in a 7% CO.sub.2 humidified atmosphere.
The blood is then centrifuged and the supernatant fluid is collected and
assayed within approximately 24 hours for reactive antibodies by ELISA
and/or Western blot techniques. In the alternative, an aliquot of fluid
may be taken directly from the sample. Each sample should be screened for
antibody by ELISA first, samples considered positive may then be subjected
to Western blot analysis.
It is to be understood that the present invention can be used to detect
antibodies in a wide variety of conditions, including, but not limited to,
antibodies to foreign antigens involved in transplantation of organs and
tissues. Supernatants of the blood from a potential recipient can be
prepared and treated according to the present invention and then stored so
that when a potential donor is found, the PBMC from the donor is mixed
with the supernatant fluid from the recipient. The presence and level of
antibodies that bind to the donor's cells can be measured by a variety of
different means including but not limited to lysis by complement or by
differential staining and FACS analysis. These methods of detecting
antibodies are well known to those of ordinary skill in the art.
The present invention can be used to determine if "hidden" antibodies are
present after or during infection by a microorganism including, but not
limited to, yeasts, bacteria, viruses, protozoa, and other classes of
microorganisms.
This invention is further illustrated by the following examples, which are
not to be construed in any way as imposing limitations upon the scope
thereof. On the contrary, it is to be clearly understood that resort may
be had to various other embodiments, modifications, and equivalents
thereof which, after reading the description herein, may suggest
themselves to those skilled in the art without departing from the spirit
of the present invention and/or the scope of the appended claims.
EXAMPLE 1
Blood from 10 different patients was analyzed for the presence of HIV
specific antibodies. In this example, the blood was collected from the
patients, and PBMC were isolated from the blood. For each patient, whole
blood and the PBMC were incubated in a medium containing pokeweed mitogen.
For whole blood, 0.4 ml of whole blood was mixed with 2 mls of culture
medium. The medium was a 1:500 final dilution of PWM in RPMI 1640
supplemented with penicillin (100 units per ml), streptomycin (100
.mu.g/ml), 2 mM L-glutamine, and 10% (v/v heat-inactivated (56.degree. C.,
30 min) fetal calf serum (all from GIBCO). For PBMC, the cells were
incubated in the same medium at a concentration of 2.times.10.sup.6 cells
per mi. In addition, serum from each patient was analyzed for the presence
of HIV antibodies. The cultures of whole blood and PBMC were performed in
triplicate in sterile test tubes and incubated for 4 days at 37.degree. C.
in a 7% CO.sub.2 humidified atmosphere. The cultures were then
centrifuged, and the supernatant fluid was collected and assayed within 24
hours for HIV-1-reactive antibodies by ELISA and PCR tests. The ELISA
tests used were purchased from Abbott Laboratories. The results are as
follows:
______________________________________
Patient Blood PBMC Serum PCR
______________________________________
1 - - - -
2 - - - -
3 - - - -
4 - - - -
5 1.2-1.5* 1.2-1.4* - +
6 - - - -
7 - - - -
8 - - - -
9 + + + +
10 + + + +
______________________________________
*O.D. reading that is "low positive" in a seronegative patient that prove
to be positive by PCR. Thus, whole blood was just as good as PBMC for
detecting HIVreactive antibodies after incubation with pokeweed mitogen.
Example II
Blood from four different monkeys was analyzed for the presence of SIV
specific antibodies. In this example, the blood was collected from the
monkeys, and PBMC were isolated from the blood. The plasma from each
sample was collected for serology after centrifugation. For each monkey,
whole blood and the PBMC were incubated in a medium containing pokeweed
mitogen. For whole blood, 0.4 ml of whole blood was mixed with 2 mls of
culture medium. The medium was a 1:500 final dilution of PWM in RPMI 1640
supplemented with penicillin (100 units per ml), streptomycin (100
.mu.g/ml), 2 mM L-glutamine, and 10% (v/v heat-inactivated (56.degree. C.,
30 min) fetal calf serum (all from GIBCO). For PBMC, the cells were
incubated in the same medium at a concentration of 2.times.10.sup.6 cells
per ml. In addition, serum from each monkey was analyzed for the presence
of SIV antibodies. The cultures of whole blood and PBMC were performed in
triplicate in sterile test tubes and incubated for 4 days at 37.degree. C.
in a 7% CO.sub.2 humidified atmosphere. The culture centrifuged, and the
supernatant fluid was collected and assayed within 24 hours for
SIV-1-reactive antibodies by ELISA and Western Blot tests. The results are
as follows:
______________________________________
Monkey Serum PBMC Whole Blood
______________________________________
Mangaby + 0.521 (++) 0.869 (++)
Mangaby - 0.211 (+) 0.351 (+)
Rhesus + 0.469 (++) 0.1208 (++)
Rhesus - 0.157 (-) 0.208 (-)
______________________________________
Example III
Blood from eight different monkeys was analyzed for the presence of SIV
specific antibodies. In this example, the blood was collected from the
monkeys, and PBMC were isolated from the blood. The plasma from each
sample was collected for serology after centrifugation. For each monkey,
whole blood and the PBMC were incubated in a medium containing pokeweed
mitogen. For whole blood, 0.4 ml of whole blood was mixed with 2 mls of
culture medium. The medium was a 1:500 final dilution of PWM in RPMI 1640
supplemented with penicillin (100 units per ml), streptomycin (100
.mu.g/ml), 2 mM L-glutamine, and 10% (v/v heat-inactivated (56.degree. C.,
30 rain) fetal calf serum (all from GIBCO). For PBMC, the cells were
incubated in the same medium at a concentration of 2.times.10.sup.6 cells
per ml. In addition, serum from each monkey was analyzed for the presence
of SIV antibodies. The cultures of whole blood and PBMC were performed in
triplicate in sterile test tubes and incubated for 4 days at 37.degree. C.
in a 7% CO.sub.2 humidified atmosphere. The cultures were then
centrifuged, and the supernatant fluid was collected and assayed within 24
hours for SIV-1-reactive antibodies by ELISA and Western Blot tests. The
results are as follows:
______________________________________
O.D. at 405 mm
Animal Blood PBMC Serum
______________________________________
G 1.059 0.0620 + low
H 0.390 0.110 -
200 0.869 0.468 + low
315 0.939 0.491 + low
2h 0.585 0.146 -
PBi 1.481 0.867 + low
neg. control
0.407 .+-. 0.070
0.120 .+-. 0.014
+ low
cut-off 0.547 0.148
______________________________________
Example IV
Blood from eight different monkeys was analyzed for the presence of SIV
specific antibodies. In this example, the blood was collected from the
monkeys, and PBMC were isolated from the blood. The plasma from each
sample was collected for serology after centrifugation. For each monkey,
whole blood and the PBMC were incubated in a medium containing pokeweed
mitogen. For whole blood, 0.4 ml of whole blood was mixed with 2 mls of
culture medium. The medium was a 1:500 final dilution of PWM in RPMI 1640
supplemented with penicillin (100 units per ml), streptomycin (100
.mu.g/ml), 2 mM L-glutamine, and 10% (v/v heat-inactivated (56.degree. C.,
30 min) fetal calf serum (all from GIBCO). For PBMC, the cells were
incubated in the same medium at a concentration of 2.times.10.sup.6 cells
per mi. In addition, serum from each monkey was analyzed for the presence
of SIV antibodies. The cultures of whole blood and PBMC were performed in
triplicate in sterile test tubes and incubated for 4 days at 37.degree. C.
in a 7% CO.sub.2 humidified atmosphere. The cultures were then
centrifuged, and the supernatant fluid was collected and assayed within 24
hours for SIV-1-reactive antibodies by ELISA and Western Blot tests. The
results are as follows:
______________________________________
Animal PBMC Blood
______________________________________
1 + +
2 + +
3 + +/-
4 + +
5 - N.D.
6 - -
7 - -
8 + N.D.
______________________________________
As can be seen from the data, there is excellent correlation between PBMC
and whole blood.
It should be understood, of course, that the foregoing relates only to a
preferred embodiment of the present invention and that numerous
modifications or alterations may be made therein without departing from
the spirit and the scope of the invention as set forth in the appended
claims.
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